CN103834573B - A kind of Fusarium oxysporum and in the degraded application of continuous cropping obstacle in poisonous substance matter - Google Patents
A kind of Fusarium oxysporum and in the degraded application of continuous cropping obstacle in poisonous substance matter Download PDFInfo
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- CN103834573B CN103834573B CN201310754556.5A CN201310754556A CN103834573B CN 103834573 B CN103834573 B CN 103834573B CN 201310754556 A CN201310754556 A CN 201310754556A CN 103834573 B CN103834573 B CN 103834573B
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- Prior art keywords
- fusarium oxysporum
- poisonous substance
- styracin
- substance matter
- continuous cropping
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- 241000223221 Fusarium oxysporum Species 0.000 title claims abstract description 49
- 239000000126 substance Substances 0.000 title claims abstract description 43
- 231100000614 poison Toxicity 0.000 title claims abstract description 40
- 230000007096 poisonous effect Effects 0.000 title claims abstract description 40
- 238000009335 monocropping Methods 0.000 title claims abstract description 27
- NQBWNECTZUOWID-UHFFFAOYSA-N (E)-cinnamyl (E)-cinnamate Natural products C=1C=CC=CC=1C=CC(=O)OCC=CC1=CC=CC=C1 NQBWNECTZUOWID-UHFFFAOYSA-N 0.000 claims abstract description 60
- NQBWNECTZUOWID-QSYVVUFSSA-N cinnamyl cinnamate Chemical compound C=1C=CC=CC=1\C=C/C(=O)OC\C=C\C1=CC=CC=C1 NQBWNECTZUOWID-QSYVVUFSSA-N 0.000 claims abstract description 60
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 claims abstract description 36
- 239000002689 soil Substances 0.000 claims abstract description 23
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 claims abstract description 18
- 235000013305 food Nutrition 0.000 claims abstract description 18
- MWOOGOJBHIARFG-UHFFFAOYSA-N vanillin Chemical compound COC1=CC(C=O)=CC=C1O MWOOGOJBHIARFG-UHFFFAOYSA-N 0.000 claims abstract description 18
- FGQOOHJZONJGDT-UHFFFAOYSA-N vanillin Natural products COC1=CC(O)=CC(C=O)=C1 FGQOOHJZONJGDT-UHFFFAOYSA-N 0.000 claims abstract description 18
- 235000012141 vanillin Nutrition 0.000 claims abstract description 18
- 235000015097 nutrients Nutrition 0.000 claims description 33
- 239000002068 microbial inoculum Substances 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 12
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical group OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 5
- 230000015556 catabolic process Effects 0.000 abstract description 38
- 238000006731 degradation reaction Methods 0.000 abstract description 38
- LPNBBFKOUUSUDB-UHFFFAOYSA-N p-toluic acid Chemical compound CC1=CC=C(C(O)=O)C=C1 LPNBBFKOUUSUDB-UHFFFAOYSA-N 0.000 abstract description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 14
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 9
- 229910052799 carbon Inorganic materials 0.000 abstract description 9
- 230000012010 growth Effects 0.000 abstract description 9
- 238000000034 method Methods 0.000 abstract description 8
- 230000033558 biomineral tissue development Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 34
- 229910017053 inorganic salt Inorganic materials 0.000 description 20
- 240000008067 Cucumis sativus Species 0.000 description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- 238000004659 sterilization and disinfection Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
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- 230000001954 sterilising effect Effects 0.000 description 6
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 229930187593 rose bengal Natural products 0.000 description 5
- AZJPTIGZZTZIDR-UHFFFAOYSA-L rose bengal Chemical compound [K+].[K+].[O-]C(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 AZJPTIGZZTZIDR-UHFFFAOYSA-L 0.000 description 5
- 229940081623 rose bengal Drugs 0.000 description 5
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- 238000012360 testing method Methods 0.000 description 5
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- 230000009102 absorption Effects 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 2
- 244000241235 Citrullus lanatus Species 0.000 description 2
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 2
- 244000241257 Cucumis melo Species 0.000 description 2
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 244000061458 Solanum melongena Species 0.000 description 2
- 235000002597 Solanum melongena Nutrition 0.000 description 2
- 244000061456 Solanum tuberosum Species 0.000 description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000035508 accumulation Effects 0.000 description 2
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- 239000008346 aqueous phase Substances 0.000 description 2
- 239000003610 charcoal Substances 0.000 description 2
- XOYLJNJLGBYDTH-UHFFFAOYSA-M chlorogallium Chemical compound [Ga]Cl XOYLJNJLGBYDTH-UHFFFAOYSA-M 0.000 description 2
- 229930016911 cinnamic acid Natural products 0.000 description 2
- 235000013985 cinnamic acid Nutrition 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
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- 238000011161 development Methods 0.000 description 2
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- 239000008103 glucose Substances 0.000 description 2
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- 238000004519 manufacturing process Methods 0.000 description 2
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- 230000008635 plant growth Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
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- 230000005477 standard model Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 230000006820 DNA synthesis Effects 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 108010029541 Laccase Proteins 0.000 description 1
- 241000209094 Oryza Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- CUBCNYWQJHBXIY-UHFFFAOYSA-N benzoic acid;2-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=CC=C1.OC(=O)C1=CC=CC=C1O CUBCNYWQJHBXIY-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- MYFXBBAEXORJNB-UHFFFAOYSA-N calcium cyanamide Chemical compound [Ca+2].[N-]=C=[N-] MYFXBBAEXORJNB-UHFFFAOYSA-N 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000002361 compost Substances 0.000 description 1
- 238000003967 crop rotation Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000009422 growth inhibiting effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
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- 238000002386 leaching Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 239000006151 minimal media Substances 0.000 description 1
- 229940045641 monobasic sodium phosphate Drugs 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229940049547 paraxin Drugs 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- -1 phenolic acid compound Chemical class 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of Fusarium oxysporum and in the degraded application of continuous cropping obstacle in poisonous substance matter.Does is the deposit number of described Fusarium oxysporum CCTCC? No.M2013432.Fusarium oxysporum of the present invention to utilize from poisonous substance matter as sole carbon source and energy growth, belongs to mineralization, to remove in cultivation water body or soil from poisonous substance matter, overcome continuous cropping obstacle there is positive meaning.When to grow 72h from poisonous substance matter for sole carbon source and the energy, 99.53% is up to the degradation rate of styracin, the degradation rate of para Toluic Acid is up to 97.9%, 92.5% is up to the degradation rate of vanillin food grade,1000.000000ine mesh, 95.6% is up to the degradation rate of P-hydroxybenzoic acid, and any side effect can not be produced to crop in degraded while poisonous substance matter; Using method is simple, convenient, with low cost, has broad application prospects.
Description
Technical field
The invention belongs to the development and utilization field of microorganism germ plasma resource, particularly relate to a kind of Fusarium oxysporum and in the degraded application of continuous cropping obstacle in poisonous substance matter.
Background technology
Refer to that plant is by overground part leaching, root system secretion and the release of the approach such as plant stubble decomposition and to same stubble or lower stubble is of the same race or equal plant-growth produces inhibiting chemical substance from poisonous substance matter.What accumulate in cucumber soil plants phenolic acid compound formation primarily of styracin, phenylformic acid and Whitfield's ointment interior more than ten from poisonous substance matter.These materials affect plant-growth by affecting the number of ways such as ionic absorption, moisture absorption, photosynthesis, protein and DNA synthesis on the one hand, and what is more important provides abundant nutrition from the rhizosphere pathogenic bacteria that is accumulated as of poisonous substance matter in rhizosphere soil, accelerate its breeding, thus cause the generation of continuous cropping obstacle.
, more easily there is the crops such as the cucumber of continuous cropping obstacle, watermelon, muskmelon, tomato and eggplant and all there is the ability of secretion from poisonous substance matter in current discovery.In recent years, due to the appearance of the management mode such as industrialization, specialization, mass-producing of industrialized agriculture, also make China various places in succession emerge the characteristic professional place of production and be engaged in the peasant of a certain specific Vegetable produce.Long-term continuous cropping causes soil from a large amount of accumulations of poisonous substance matter, and many continuous cropping soils need the crop rotation of more than 5 years to alleviate continuous cropping obstacle.For the manager of professional production, become the important factor causing continuous cropping obstacle, restriction industry development, restriction to increase income from the accumulation of poisonous substance matter.
The most directly remove and comprise the physico-chemical method such as charcoal absorption and applying soil disinfection material (such as lime nitrogen) from the method for poisonous substance matter.But charcoal absorption is only applicable to the soilless culture such as water planting, substrate culture, is reached from the purification of poisonous substance matter by the filtration of gac in the rich water recycle system, cannot be applied in soil.And soil disinfection is because of high cost, can be limited in actual applications reasons such as crop growth are unfavorable environment and excessive using.Seek low cost, the task of top priority that high-level efficiency, the removal that has no side effect from the method for poisonous substance matter are researcher.
As everyone knows, soil has self-repairing capability, this is because the microorganism survived in soil can secrete the materials such as peroxidase, laccase, lytic enzyme, and these materials all can decompose the chemical substance in environment.Therefore screening, being separated efficient degradation continuous cropping obstacle to be remove the optimization approach from poisonous substance matter from the microorganism of poisonous substance matter.
Publication No. is that the Chinese patent literature of CN103045506A discloses the composite microbiological fertilizer of a kind of continuous cropping obstacle from poisonous substance matter degradation bacteria and preparation thereof, and this continuous cropping obstacle is subtilis from poisonous substance matter degradation bacteria, and deposit number is CGMCC No.6896.Whether this bacterial strain para Toluic Acid has certain degradation capability, but does not record the degradation efficiency of this bacterial strain para Toluic Acid, and have Degradation also not known from poisonous substance matter beyond this bacterial strain para Toluic Acid, cannot effectively eliminate the continuous cropping obstacle caused from poisonous substance matter.
Summary of the invention
The invention discloses a kind of Fusarium oxysporum, this bacterial strain to effectively degrading from poisonous substance matter of existing in cultivation matrix, can eliminate continuous cropping obstacle.
A kind of Fusarium oxysporum, Classification And Nomenclature is Fusarium oxysporum (Fusarium oxysporum), strain number is YY186-1, be preserved in the China typical culture collection center (CCTCC) being positioned at Luo Jia Shan, Wuhan City Wuhan University on September 17th, 2013, deposit number is CCTCC No.M2013432.
The 18SrRNA sequence of this Fusarium oxysporum is as shown in SEQ ID No.1, and its main biological property is: bacterium colony projection is cotton-shaped, and white powder, matter are close; Spore opaque, sickleshaped, bend a little, majority be 3 every; Optimum growing condition is pH value 7.0, temperature 28 DEG C, and can using styracin, phenylformic acid, vanillin food grade,1000.000000ine mesh or P-hydroxybenzoic acid as sole carbon source and energy growth.
Based on the growth characteristics of this bacterial strain, present invention also offers the growth medium of a kind of described Fusarium oxysporum, comprise carbon source and inorganic salt, described carbon source is at least one in styracin, phenylformic acid, vanillin food grade,1000.000000ine mesh or P-hydroxybenzoic acid.
As preferably, described carbon source is styracin, and concentration is 75 ~ 150mg/L.In styracin, phenylformic acid, vanillin food grade,1000.000000ine mesh or P-hydroxybenzoic acid, the utilization ratio of this Fusarium oxysporum to styracin is the highest.
As preferred further, the formula of described growth medium is: styracin 150mg/L, NaCl0.5g, MgSO
4.7H
2o0.2g, NH
4nO
31.0g, KHPO
41.0g, K
2hPO
41.0g, GaCl
22H
2o0.02g, FeCl
36H
2o0.05g, distilled water complements to 1000mL, stirs, adjust ph to 7.0, high pressure steam sterilization 20min at 121 DEG C.
Fusarium oxysporum of the present invention is separated from continuous cropping soil, is to obtain through the Selective agar medium screening containing styracin.Styracin is more representative from poisonous substance matter in continuous cropping soil, therefore present invention also offers described Fusarium oxysporum in the degraded application of continuous cropping obstacle in poisonous substance matter, described application comprises: in the nutrient solution that Fusarium oxysporum bacteria suspension is added to planting plants or soil.
Because Fusarium oxysporum of the present invention to utilize from poisonous substance matter as sole carbon source and energy growth, belong to mineralization, to remove in cultivation water body or soil from poisonous substance matter, overcome continuous cropping obstacle there is positive meaning.
Described continuous cropping obstacle continuous cropping obstacle is phenylformic acid, vanillin food grade,1000.000000ine mesh, P-hydroxybenzoic acid or styracin from poisonous substance matter.
After tested, when (adding bacterium liquid wild Oryza species OD in the growth medium that to be added to by Fusarium oxysporum bacteria suspension with respective poisonous substance matter be sole carbon source and the energy
415be about 0.2) time, the degradation rate of Fusarium oxysporum to styracin is up to 99.53%, and the degradation rate of para Toluic Acid is up to 97.9%, is up to 92.5% to the degradation rate of vanillin food grade,1000.000000ine mesh, is up to 95.6% to the degradation rate of P-hydroxybenzoic acid.
Present invention also offers a kind of microbial inoculum including described Fusarium oxysporum, the OD of this microbial inoculum
415be 2.0 ~ 3.0.This bacteria suspension can be the aqua made after being mixed with conventional additives by described Fusarium oxysporum, and described additives can select the phosphoric acid buffer (pH7.0) of 0.2 ~ 0.3mol/L.
At needs to when carrying out degradation treatment from poisonous substance matter, described microbial inoculum is directly added (or adding after suitable multiple dilution) in the nutrient solution or soil of planting plants, as preferably, after described microbial inoculum is diluted ten times, be added in nutrient solution with the ratio of 5 ~ 15mL/10L, or with 0.5-2L/m
2ratio be added in soil.
As preferably, the interpolation of microbial inoculum is opportunity: for life-time service, nutrient solution that replacement frequency is lower, preferably add microbial inoculum at the intermediate period of crop growth period; For flowing nutrient solution then without the need to adding microbial inoculum; For earth culture, if the preceding crop crop that to be continuous cropping obstacle more serious, preferably microbial inoculum was added in soil in 1-15 days before second stubble crop field planting, then without the need to adding microbial inoculum in the vegetative period of second stubble crop.
In the water body that Fusarium oxysporum of the present invention can be used for planting the various crop such as cucumber, watermelon, muskmelon, tomato, eggplant or soil, as long as these crops can secrete at least one in phenylformic acid, vanillin food grade,1000.000000ine mesh, P-hydroxybenzoic acid or styracin from poisonous substance matter.
Compared with prior art, beneficial effect of the present invention is embodied in:
Fusarium oxysporum of the present invention can degrade fast, efficient, safely crop secretion in water body and soil from poisonous substance matter, when to grow 72h from poisonous substance matter for sole carbon source and the energy, 99.53% is up to the degradation rate of styracin, the degradation rate of para Toluic Acid is up to 97.9%, 92.5% is up to the degradation rate of vanillin food grade,1000.000000ine mesh, 95.6% is up to the degradation rate of P-hydroxybenzoic acid, and any side effect can not be produced to crop in degraded while poisonous substance matter; Using method is simple, convenient, with low cost, has broad application prospects.
Accompanying drawing explanation
Fig. 1 is the flat-plate bacterial colony growth figure of Fusarium oxysporum of the present invention;
Fig. 2 is the conidium microscopic morphology observation figure of Fusarium oxysporum of the present invention;
Fig. 3 is the degradation curve of Fusarium oxysporum of the present invention para Toluic Acid under pure culture condition;
Fig. 4 is Fusarium oxysporum of the present invention degradation curve to vanillin food grade,1000.000000ine mesh under pure culture condition;
Fig. 5 is Fusarium oxysporum of the present invention degradation curve to P-hydroxybenzoic acid under pure culture condition;
Fig. 6 applies Fusarium oxysporum of the present invention to the impact of cucumber plant dry weight.
Embodiment
1 substratum and solution preparation
(1) inorganic salt nutrient solution: NaCl0.5g, MgSO
47H
2o0.2g, NH
4nO
31.0g, KHPO
41.0g, K
2hPO
41.0g, GaCl
22H
2o0.02g, FeCl
36H
2o0.05g, distilled water supplies 1000mL, stirs, adjust ph to 7.0, high pressure steam sterilization 20min at 121 DEG C.
(2) beef-protein medium: extractum carnis 10g, peptone 5.0g, sodium-chlor 5.0g, distilled water complement to 1000mL, stir after mixing, adjust ph to 7.0, high pressure steam sterilization 20min at 121 DEG C.
(3) rose bengal medium: KH
2pO
41.0g, MgSO
47H
2o0.5g, peptone 5g, glucose 5g, agar 16g, rose-bengal 0.053g, distilled water 1000mL, adjust ph to 7.0, high pressure steam sterilization 20min at 121 DEG C; The paraxin of 30 μ g/mL is added before using.
(4) PDA substratum: potato is cleaned peeling, then takes 200g potato and be cut into small pieces, adds water and well-donely (boils 20 ~ 30 minutes, can be poked by glass stick), by eight layers of filtered through gauze, heating, add agar 15g/L, continue heated and stirred mixing, after agar has dissolved, add glucose 20g, stir, slightly supply moisture to 1000 milliliter again after cooling, at 121 DEG C, high pressure steam sterilization took out after 20 minutes, and after cooling, storage is for subsequent use.
(5) 0.2mol/L phosphoric acid buffer (pH7.0): Sodium phosphate dibasic (2mol/L) 61mL, SODIUM PHOSPHATE, MONOBASIC (2mol/L) 39mL, adding distil water is settled to 1000mL, high pressure steam sterilization 20min at 121 DEG C.
2 strains separation purifying
(1) continuous cropping soil sample picks up from farm, Zhejiang University Hua Jiachi school district, gets 1g continuous cropping soil and is placed in 100mL triangular flask, adds 20mL inorganic salt nutrient solution, dark shaking culture (28 DEG C, 180r/min) 1 week;
(2) getting the turbid liquid 1mL in upper strata is inoculated in the inorganic salt nutrient solution containing 75mg/L styracin, dark shaking culture (28 DEG C, 180r/min) 1 week;
(3) repeating step (2) 4 times, when wherein cultivating for 1st ~ 3 times, in inorganic salt nutrient solution, styracin concentration is 75mg/L, when the 4th is cultivated, in inorganic salt nutrient solution, styracin concentration is 100mg/L, and each inoculum cultivated all takes from the nutrient solution cultivating gained last time;
(4) dip a small amount of the 4th and cultivate the nutrient solution obtained, is being separated containing beef extract-peptone and the rose bengal medium flat board of 100mg/L styracin carry out line respectively, in the middle cultivation of biochemical cultivation case (28 DEG C);
(5) after the single bacterium colony of dull and stereotyped upper appearance, single bacterium colony that picking feature is different is switched in the inorganic salt nutrient solution containing 100mg/L styracin and carries out shaking table cultivation (28 DEG C, 180r/min) 1 week;
(6) from every part of nutrient solution of step (5), dip a small amount of nutrient solution be more respectively transferred in new 100mg/L styracin inorganic salt nutrient solution and carry out shaking table cultivation (28 DEG C, 180r/min) 1 week;
(7) repeating step (6) 5 times, chooses the bacterial strain that still can grow fast in the inorganic salt nutrient solution containing 100mg/L styracin, is inoculated on rose-bengal slant medium, preserves.
3 identification of strains
(1) identification of morphology
Rule on PDA substratum by bacterial strain picking list spot on rose-bengal slant medium, cultivate 3 days at 28 DEG C, bacterium colony projection is cotton-shaped, white powder, matter close (Fig. 1); Picking mycelia makes water slide and carries out microscopic examination, and as shown in Figure 2, spore opaque, sickleshaped, bend observations a little, majority be 3 every.
(2) Molecular Identification
Adopt regular-PCR method extract on this strain ribosome DNA ITS sequence, and entrust Nanjing Genscript Biotechnology Co., Ltd. to check order, sequencing result is as shown in SEQ ID No.1.NCBI carries out sequence alignment and analysis, is Fusarium oxysporum (Fusarium oxysporum) by this identification of strains.
4 microbial inoculum preparations
(1) strain inoculation be deposited on test tube slant is contained in the inorganic salt nutrient solution of 100mg/L styracin in 20mL, activation culture 7 days at 28 DEG C;
(2) get the bacterial classification 100 μ L activated and be inoculated in 200mL containing in the beef-protein medium of 100mg/L styracin, 28 DEG C, 180r/min shaking culture is to logarithmic phase;
(3) by centrifugal for the bacterial classification of logarithmic phase (8000 × g) 10min, outwell supernatant, the phosphoric acid buffer (pH7.0) of thalline 30mL0.2mol/L washs 3 times;
(4) thalline that step (3) obtains is suspended in the phosphoric acid buffer (pH7.0) of 0.2mol/L, makes OD
415be the bacteria suspension of 2.0 ~ 3.0, be microbial inoculum.
The detection of styracin in 5 minimal mediums
The inorganic salt nutrient solution that 20mL contains styracin is proceeded in beaker, regulates pH to 8.0 with 1mol/L sodium hydroxide, then add appropriate anhydrous diethyl ether, be placed in separating funnel thermal agitation, after stratification, collect lower floor's liquid (aqueous phase).
Regulate the pH to 2.0 of aqueous phase with 1mol/L HCl, then add appropriate anhydrous diethyl ether, vibration shakes up, and collects upper strata (organic phase); Add the addition of C aCl
2or CaSO
4dewater, and be placed in 40 DEG C of water-baths ether is volatilized; To be concentratedly to 1-2mL, be transferred to centrifuge tube high speed centrifugal (10000 × g, 10min), add dissolve with methanol again after removing ether, carry out Determination of cinnamic acid analysis with high performance liquid chromatography; Analytical parameters is:
High performance liquid chromatograph: SHIMADZU LC-10AD;
Detector: UV-detector;
Liquid-phase chromatographic column: ODS120 post (4.6 × 250nm);
Moving phase: the acetum of 2%: methyl alcohol (chromatographically pure)=1:1;
Overall flow rate: 1mL/min;
Sample size: 5 μ L;
Column temperature: 40 DEG C;
Minute: 20min;
Measure wavelength: 280nm.
The calculation formula of styracin residual quantity is as follows:
X=（A
x×V
o）/（A
o×V
x）×C
s；
Wherein: X is the concentration (mg/L) of styracin in testing sample; A
xfor the peak area of styracin in sample; A
ofor styracin standard model peak area; V
xfor sample volume (mL); V
ofor last constant volume (mL); C
sfor the concentration (mg/L) of styracin standard model.
The calculation formula of styracin degradation rate is as follows:
P（%）=（1-C
x/C
o）×100%
Wherein: P is the degradation rate (%) of styracin in testing sample; C
xfor styracin residual concentration (mg/L) in sample; C
ofor styracin starting point concentration (mg/L) in sample.
6 styracin recovery tests
Preparation contains the inorganic salt nutrient solution of 0.1,1,10 and 100mg/L styracin respectively, then according to the method for the 5th part, the styracin in minimal media liquid is reclaimed, and utilizing high performance liquid chromatograph to detect Determination of cinnamic acid, each concentration repeats 3 times, does blank test simultaneously.In inorganic salt nutrient solution, the average recovery rate of styracin and the variation coefficient are in table 1.
The average recovery rate of styracin and the variation coefficient in table 1 inorganic salt nutrient solution
| Add concentration (mg/L) | Sample size (mL) | Average recovery rate (%) | The variation coefficient (%) |
| 0.1 | 20 | 98.0 | 3.2 |
| 1 | 20 | 99.3 | 2.5 |
| 10 | 20 | 96.8 | 1.1 |
| 100 | 20 | 97.2 | 4.7 |
As shown in Table 1, the average recovery rate of styracin in inorganic salt nutrient solution is 96.8% ~ 99.3%, and the variation coefficient is 1.1% ~ 4.7%.Show that the detection method of styracin in above-mentioned inorganic salt nutrient solution meets the requirement from malicious species analysis.
7 pure culture Fusarium oxysporums are to the degraded of respective poisonous substance matter
(1) to the degraded of styracin
In the 100mL triangular flask that 20mL inorganic salt nutrient solution is housed, add styracin be respectively 75mg/L, 150mg/L to final concentration; Microbial inoculum prepared by the 4th part is inoculated in the inorganic salt nutrient solution containing styracin to nutrient solution OD
415be 0.2, this is experimental group; Arrange control group simultaneously, do not inoculate microbial inoculum in control group, experimental group and control group respectively establish three repetitions; Then triangular flask is placed in shaking table (28 DEG C, 180r/min) dark shaking culture.
Timing sampling when dark shaking culture 24h, 48h, 72h, detects the residual quantity of styracin in nutrient solution according to the method for the 5th part, calculate Fusarium oxysporum to the degradation rate of styracin, calculation result is in table 2.
Table 2 Fusarium oxysporum is to the degradation rate (%) of styracin
As shown in Table 2, cultivate Fusarium oxysporum after 72 hours 75mg/L and 150mg/L styracin is almost thoroughly degraded, its degradation rate is respectively 98.99% and 99.53%, and do not inoculate in the control group of Fusarium oxysporum, after 72 hours, the degradation rate of styracin is 14.77% ~ 34.04%, shows that Fusarium oxysporum has very strong degradation capability to styracin.
(2) degraded of para Toluic Acid, vanillin food grade,1000.000000ine mesh and P-hydroxybenzoic acid
In the 100mL triangular flask that 20mL inorganic salt nutrient solution is housed, add phenylformic acid, vanillin food grade,1000.000000ine mesh and P-hydroxybenzoic acid to final concentration is respectively that in 75mg/L(Fig. 3, annotation is 0.5mmol/L, inconsistent, please unify), microbial inoculum prepared by the 4th part is inoculated in the inorganic salt nutrient solution containing styracin to nutrient solution OD
415be 0.2, this is experimental group; Arrange control group simultaneously, do not inoculate microbial inoculum in control group, experimental group and control group respectively establish three repetitions; Then triangular flask is placed in shaking table (28 DEG C, 180r/min) dark shaking culture.
Timing sampling when dark shaking culture 24h, 48h, 72h, the residual quantity of phenylformic acid in nutrient solution, vanillin food grade,1000.000000ine mesh and P-hydroxybenzoic acid is detected respectively according to the method for the 5th part, calculate the degradation rate of Fusarium oxysporum para Toluic Acid, vanillin food grade,1000.000000ine mesh and P-hydroxybenzoic acid, calculation result is shown in Fig. 3, Fig. 4, Fig. 5.
From Fig. 3, Fig. 4, Fig. 5, Fusarium oxysporum para Toluic Acid, vanillin food grade,1000.000000ine mesh and P-hydroxybenzoic acid all have stronger degradation capability.Wherein para Toluic Acid degradation rate the highest, reach 97.9%(72h); Taking second place to the degradation rate of vanillin food grade,1000.000000ine mesh, is 92.5%(72h); Relatively low to the degradation rate of P-hydroxybenzoic acid, but also reach 95.6%(72h).
In control group, want high from the natural degradation rate of poisonous substance matter relative to styracin for these three kinds, wherein the natural degradation rate of vanillin food grade,1000.000000ine mesh reaches 63.6%(72h), benzoic natural degradation rate is 14.3%(72h), the rate of being degraded of P-hydroxybenzoic acid is 52.7%(72h).
Wherein, containing phenolic hydroxyl group in the chemical structure of vanillin food grade,1000.000000ine mesh and P-hydroxybenzoic acid, easily oxidized, can natural degradation under light and when humidity is larger.
8 Fusarium oxysporums alleviate the growth-inhibiting effect that styracin causes
Cucumber " is ground No. 4, Tianjin " planting seed, in soil, moves into after two weeks and is equipped with in the earthen basin of 10L compost and waters ten times of diluent (OD of 50mL fusarium oxysporum inoculant
415be 0.2); Within 2 days, water the styracin solution that 500mL concentration is 0.25mM afterwards, within 10 days, gather the root of cucumber plant, stem and leaf afterwards and to complete in an oven respectively post-drying weighing, weighing results is shown in Fig. 6.
As seen from Figure 6, with cucumber plant (the root dry weight 0.23g of health, the heavy 0.63g of stem, leaf dry weight 2.14g) compare, apply the cucumber plant of styracin, its root dry weight (0.11g), stem heavy (0.36g) and leaf dry weight (1.04g) are all decreased significantly, and show that the growth of styracin to cucumber plant is inhibited.And compared with applying the cucumber plant of styracin, apply the cucumber plant of styracin and Fusarium oxysporum simultaneously, its root dry weight (0.23g), stem heavy (0.51g) and leaf dry weight (1.69g) rise all to some extent, show that Fusarium oxysporum can obviously be alleviated from poisonous substance matter styracin the detrimentally affect of cucumber growth.
Claims (6)
1. a Fusarium oxysporum, is characterized in that, called after Fusarium oxysporum (Fusariumoxysporum) YY186-1, and deposit number is CCTCC No.M 2013432.
2. one kind includes the microbial inoculum of Fusarium oxysporum as claimed in claim 1.
3. microbial inoculum as claimed in claim 2, is characterized in that, the OD of described microbial inoculum
415be 2.0 ~ 3.0.
4. as claimed in claim 1 Fusarium oxysporum in the degraded application of continuous cropping obstacle in poisonous substance matter.
5. apply as claimed in claim 4, it is characterized in that, comprising: in the nutrient solution that Fusarium oxysporum bacteria suspension is added to planting plants or soil.
6. apply as claimed in claim 4, it is characterized in that, described continuous cropping obstacle is phenylformic acid, vanillin food grade,1000.000000ine mesh, P-hydroxybenzoic acid or styracin from poisonous substance matter.
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| 不同抗连作障碍品种草莓根系分泌物化感物质差异分析及其化感效应;赵绪生等;《河北农业大学学报》;20120531;第35卷(第3期);第100-105页 * |
| 酚酸类物质和氨基酸对西瓜专化型镰刀菌生长的影响;刘爱民等;《湖北农业科学》;20111031;第50卷(第20期);第4181-4184页 * |
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